1. Cloning and characterization of the novel d-aspartyl endopeptidase, paenidase, from Paenibacillus sp. B38
Satoru Nirasawa, Kazuhiko Nakahara, Saori Takahashi J Biochem. 2018 Aug 1;164(2):103-112. doi: 10.1093/jb/mvy033.
Paenidase is the first micro-organism-derived d-aspartyl endopeptidase that specifically recognizes an internal d-Asp residue to cleave [d-Asp]-X peptide bonds. Using peptide sequences obtained from the protein, we performed PCR with degenerate primers to amplify the paenidase I-encoding gene. Nucleotide sequencing revealed that mature paenidase I consist of 322 amino acid residues and that the protein is encoded as a pro-protein with a 197-amino-acid N-terminal extension compared to the mature protein. Paenidase I exhibits amino acid sequence similarity to several penicillin-binding proteins. In addition, paenidase I was classified into peptidase family S12 based on a MEROPS database search. Family S12 contains serine-type d-Ala-d-Ala carboxypeptidases that have three active site residues (Ser, Lys and Tyr) in the conserved motifs Ser-Xaa-Thr-Lys and Tyr-Xaa-Asn. These motifs were conserved in the primary structure of paenidase I, and the role of these residues was confirmed by site-directed mutagenesis.
2. Substrate stereoselectivity of mammalian D-aspartyl endopeptidase
Tadatoshi Kinouchi, Norihiko Fujii, Noriko Fujii J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Nov 1;879(29):3349-52. doi: 10.1016/j.jchromb.2011.08.031. Epub 2011 Aug 30.
The formation and accumulation of D-aspartate residue (D-Asp) in proteins caused by oxidative stress leads to dysfunction and/or denaturation of proteins, and is consequently responsible for aging-related misfolding diseases such as cataracts, prion disease, and Alzheimer's disease. We sought to identify that an unknown protease selectively degrades the noxious D-Asp-containing protein, namely D-aspartyl endopeptidase (DAEP), and finally purified it from the inner mitochondrial membrane of mouse liver. In order to analyze the substrate stereoselectivity of DAEP, we synthesized a peptide corresponding to 55-65 (Thr-Val-Leu-Asp-Ser-Gly-Ile-Ser-Glu-Val-Arg) of human αA-crystallin and its corresponding diastereoisomers in which L-α-Asp was replaced with L-β-, D-α- or D-β-Asp residue at position 58. Following incubation of that peptide with purified DAEP, it was only degraded at D-α-Asp(58), independent of ATP or NAD. This result indicates that DAEP stereoselectively recognizes and degrades its substrate at the internal D-α-Asp residue. DAEP therefore seems to physiologically serve as the quality control system against the noxious D-Asp-containing protein in the long life span of mammals.
3. Isolation and characterization of mammalian D-aspartyl endopeptidase
T Kinouchi, H Nishio, Y Nishiuchi, M Tsunemi, K Takada, T Hamamoto, Y Kagawa, N Fujii Amino Acids. 2007 Jan;32(1):79-85. doi: 10.1007/s00726-006-0348-4. Epub 2006 Oct 6.
The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease (AD), cataracts and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600 kDa) and is localized in the inner mitochondrial membrane. However, DAEP activity was not detected in E. coli, S. cerevisiae, and C. elegans. A specific inhibitor for DAEP, i-DAEP: (benzoyl-L-Arg-L-His-[D-Asp]-CH(2)Cl; MW: 563.01), was newly synthesized and inhibited DAEP activity (IC(50), 3 microM), a factor of ten greater than lactacystin on DAEP. On the other hand, i-DAEP did not inhibit either the 20S or 26S proteasome. And we identified succinate dehydrogenase and glutamate dehydrogenase 1 as components of DAEP by affinity label using biotinylated i-DAEP. In the long life span of mammals, DAEP may serve as a scavenger against accumulation of racemized proteins in aging. Insights into DAEP will provide the foundation for developing treatments of diseases, such as AD, in which accumulation of D-Asp-containing proteins are implicated.